Immmunoglobulins (IgGs) are large molecules. They are antibodies composed of two heavy chains and two light chains. Each light chain has two portions; a constant region (LC) and a variable region (LV). Each heavy chain has one variable portion (HV) and four constant portions (CH1, CH2, CH3 and CH4). Each variable portion on each chain is followed by a constant portion. Each light chain is linked to a heavy chain by one or more covalent interchain disulfide bond(s). Each heavy and light chain also contains regularly spaced intra-chain disulfide bridges. The variable domains are composed of complementarity determining regions (CDRs) and framework regions (FWs) that are specific to each antibody. The variable domains determine the function and binding of the antibody. CDRs are the primary binding segments of the antibody involved in antigen binding. The constant domain, composed of the CH1, CH2, and CH3 regions, is not involved in antigen binding. The CH2 and CH3 constitute the Fc region of the antibody. The Fc region is responsible for several “effector functions” (see below), including certain biological activities which can be unfavorable in the context of therapeutic neutralizing antibodies. Such unfavorable effector functions include C1q binding and antibody-dependent cellular cytotoxicity (ADCC) response. The Fc region of an antibody, specifically the effector domain, binds to Fc receptors (Fc.gamma.Rs) on the surface of immune effector cells such as macrophages. This leads to the phagocytosis or lysis of the targeted cells. In complement-dependent cytotoxicity (CDC), the antibodies kill the targeted cells by triggering the complement cascade, and resulting MAC formation, at the cell surface.
There are five major classes of immune-globulins: IgA, IgD, IgE, IgG and IgM. The IgG(s) are further divided into four isotypes: IgG1, IgG2, IgG3, and IgG4. These isotypes elicit differential responses due to the sequences located within the constant domains. The IgG1, IgG2, and IgG3 isotypes are known to cause complement system activation and CDC. (See FIG. 1). The IgG1 and IgG3 isotypes are known to mediate ADCC. The four IgG isotypes vary in the compositions of their heavy chains. Among therapeutic antibodies, human IgG1 is the most commonly used isotype. Accordingly, attempts to modify therapeutic antibody effector functions have been focused on IgG1 isotypes. However, different IgG isotypes, with different inherent effector function properties, are often more useful in the context of reducing and/or exploiting effector functions. There is a need in the art to develop the use of the IgG2, IgG3, IgG4 isotypes, and isotype hybrids, with and without Fc modifications, for use as therapeutic antibodies. The present invention aims to address some of these needs.
Hybrid Isotypes—The hinge region of the IgG antibody is located between the CH1 and CH2 portions of the heavy chain, and is particularly susceptible to proteolytic cleavage. Antibodies can be recombinantly engineered to form hybrid isotypes which contain portions from two or more different isotypes. For example, the prior art includes a hybrid antibody which contains the variable portion and the first constant portion of an IgG2 isotype antibody fused to the CH2, and CH3 portions of an IgG4 isotype antibody. (US Patent Application 20070041972).
Fc Mediated Effector Functions—The Fc “effector functions” are the biological activities of an antibody (be it a natural antibody or an engineered antibody) other than the antibody's primary function and purpose. In the case of therapeutic neutralizing antibodies, the effector functions are the biological activities of the antibody other than the neutralization of the target protein or pathway. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); lack of activation of platelets that express Fc receptor; and B cell activation. Many effector functions begin with Fc binding to an Fc gamma receptor (Fc.gamma.R). There are three subclasses of Fc.gamma.R: Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16).